Bacterial plasmids are extrachromosomal DNA molecules that replicate autonomously in the host cell. They vary in size from one to several hundred kilobases (kB) and in copy number from one to several hundred per cell. However, not all plasmids are inherently useful for a molecular biologist to use as a cloning vector. Desirable characteristics of a cloning vector include: 1.) small size (i.e., less than about 6 kB); 2.) relatively high copy number; 3.) presence of a selectable marker; and 4.) presence of single sites for cleavage by restriction enzymes.
Applications using non-enteric bacteria for basic and applied molecular research have extended the criteria one must consider with respect to choice of a plasmid cloning vector. Specifically, host range refers to the types of microbes in which a plasmid will replicate. One may develop a specific vector for each microbial species of interest, or one may take advantage of available broad host range replicons which have the ability to be maintained in a wide range of microbes that are unrelated. These broad host range plasmids typically encode all of their own proteins required for replication initiation, and therefore are not dependent on their host cell. In contrast, narrow host range replicons may lack replication or segregation proficiencies (as compared to an inability to be introduced into or express genetic markers in a distantly related host), which result in their replication only in closely related species (Schmidhauser, T. J. and D. R. Helinski. J. Bacteriol. 164:446–455 (1985)). Most broad host range plasmids are classified on the basis of their intrinsic properties, according to their “incompatibility groups”. This classification reflects the similarities in sequence, function, and the nature of the replicon (as replicons of the same type are unable to co-exist in a cell, while replicons from different incompatibility groups (e.g., “Inc” groups) may exist simultaneously in a single cell). Natural plasmid isolates of gram-negative bacteria that belong to incompatibility groups C, N, P, Q and W display replication and maintenance proficiency in a diversity of bacterial species.
A particularly useful broad host range plasmid is the commercially available pBHR1 (MoBiTec; Göttingen, Germany; GenBank Y14439). This plasmid was derived from the broad host range plasmid pBBR1, which was isolated from the gram-negative bacterium Bordetella bronchiseptica S87 (Antoine, R. and C. Locht, Mol. Microbiol. 6(13):1785–1799 (1992); FR 2,690,459). Like pBBR1, pBHR1 does not belong to any of the common broad host range incompatibility groups and possesses a relatively high copy number. Both plasmids possess two critical open reading frames (ORFs)—the first, known as rep, is involved in replication of the plasmid; and the second ORF is known as mob. The mob gene, involved in mobilization, has been extensively characterized for this family of plasmids by Szpirer et al. (Molecular Microb. 37(6): 1283–1292 (2000); J. Bacteriol. 183(6): 2101–2110 (2001)). Plasmid pBHR1 also additionally has two selectable markers (kanamycin and chloramphenicol), while maintaining a relatively small size of only 5300 bp. These properties render pBHR1 as an extremely useful cloning vector suitable for a wide range of gram-negative bacteria.
One variation that would increase the utility of pBHR1 would be creation of a temperature sensitive (Ts) mutant, such that it would be possible to control when the plasmid replicated within the host cell. Temperature sensitive mutants typically express their mutant phenotypes at elevated temperatures. Ideally, the replication Ts mutant of pBHR1 would grow and replicate normally at the permissive temperature, but the mutant phenotype (i.e., a non-replicative plasmid) would only be expressed at the elevated, or “restrictive”, temperature in a conditional manner. Growth of the bacterial host under these restrictive conditions would result in plasmid elimination or integration of the plasmid into the chromosome at regions of significant DNA homology. Thus, a Ts-pBHR1 plasmid would be useful for: 1.) plasmid curing (whereby it is desirable to eliminate a plasmid from a bacterial strain); 2.) plasmid chasing (whereby it is possible to remove, or “chase”, other plasmids from a bacterial strain that are incompatible with the subsequently introduced plasmid); and 3.) the creation of single and double crossover mutants by homologous recombination (whereby a particular region of DNA can be integrated into a host chromosome from a plasmid). It is especially desirable to have an effective means available for introducing or specifically and permanently modifying certain genes in microbial host organisms.
Temperature sensitive mutants are generated by random mutagenesis followed by screening to obtain mutants with a Ts phenotype. Although the technique of generating these mutants is well understood by an artisan skilled in molecular biology, the tremendous utility and need for development of a Ts-pBHR1 plasmid has not previously been recognized. Furthermore, it is impossible to predict which mutations in the replication control region of pBHR1 will encode temperature sensitive mutations.
The problem to be solved therefore is to develop a temperature sensitive broad host range plasmid having the ability to co-exist with a variety of other broad host range plasmids.
Applicants have solved the stated problem by isolating a suite of temperature sensitive plasmids derived from pBHR1, containing mutant replication control regions. The broad host range of the plasmid, and its compatibility with other known broad host range vectors, makes the Ts-plasmid of the present invention particularly attractive for the genetic engineering of non-enteric bacteria for basic and applied molecular research.